1. Field of the Invention
This invention relates to the field molecular biology, specifically with reference to the subject of DNA polymerases for use in the polymerase chain reaction and DNA sequencing.
2. Description of the Prior Art
Polymerase Chain Reaction (PCR) was one of the most important inventions developed in area of biotechnology during the 1980's and has proven useful for a variety of tasks. PCR Technology, Principles and Applications for DNA Amplification (Erlich ed. 1989). The process provides a method for amplifying known specific nucleic acid sequences. Mullis, U.S. Pat. No. 4,683,202. The process comprises treating single- or double-stranded DNA containing the sequence of interest with an excess of two oligonucleotide primers sufficiently complementary of the strands so as to hybridize to the denatured strands. The hybridized primers are then extended by a DNA polymerase in the presence of the four dNTPs. The primer extension products are then separated and can serve as templates for another cycle of replication. The number of DNA templates approximately doubles on each cycle of amplification. Thus, 20 cycles of the process will result in approximately a 2.sup.20 -fold amplification.
The original protocols for PCR used the Klenow fragment of E. coli DNA polymerase I to catalyze the extension of the oligonucleotide primers. Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51, 263 (1986); Mullis and Faloona, Methods Enzymol. 155, 335 (1987). The Klenow fragment proved somewhat cumbersome to use. Denaturation of the double stranded DNA at the start of each cycle requires temperatures ranging from 80.degree. to 105.degree. C. These temperatures inactivate the Klenow fragment. Consequently, fresh enzyme was required at the start of each new amplification cycle. While this process generally worked well for small segments of DNA (&lt;200 bp), a host of problems arose when replication of larger fragments was attempted.
The difficulties associated with use of the Klenow fragment DNA polymerase were circumvented with the introduction of thermostable DNA polymerase obtained from the thermophilic bacterium Therrnus aquaticus (Taq DNA polymerase). Saiki et al., Science 239, 487 (1989); Gelfand et al., U.S. Pat. No. 4,889,818. This enzyme has been cloned, overproduced, and the DNA sequence determined. Lawyer et al., J. Biol. Chem. 264, 6427-6437 (1989).
In addition to its DNA polymerase activity, Taq DNA polymerase also possesses 5'-3' polymerization-dependent exonuclease activity, but it lacks 3'-5' exonuclease activity. Longley et al., Nuc. Acids Res. 18, 7317-7322 (1990); Blanco et al., Gene 100, 27-38 (1991); Bernad et al., Cell 59, 219-228 (1989); Lawyer et al., supra; Holland et al., Proc. Natl Acad. Sci. 88, 7276-7280 (1991); and Kelly and Joyce, J. Mol. Biol. 164, 529-560 (1983). Studies have identified the 5'-3' exonuclease activity as being an intrinsic part of Taq DNA polymerase. Longely et al., supra; and Barnes et al., Gene 112, 29-35 (1992). This activity appears to facilitate a nick translation DNA reaction.
Native Taq DNA polymerase suffers from a high rate of misincorporation--about four times higher than that of the Klenow fragment of E. coli DNA polymerase I. It has been estimated that Taq DNA polymerase incorporates one incorrect nucleotide in 9000. Tindall and Kunkel, Biochemistry 27, 6008 (1988). After 20 amplification cycles, this would result in DNA molecules with random mutations averaging one in every 900 bases. Saiki et al., supra. If the PCR product is to be inserted into an expression vector, the chance that one cloned molecule will contain an unwanted sequence alteration may be significant. It would be desirable, therefore, to decrease the rate of misincorporation of the DNA polymerase used in PCR without sacrificing the heat stability and rate of synthesis of the native Taq DNA polymerase.
It has been shown that removal of the 5'-most 235 codons of the Taq DNA polymerase gene results in an expression product that has no 5'-3' exonuclease activity and a lower rate of mutagenesis. Tindall et al., supra; and Barnes, supra.
Other forms of Taq DNA polymerase are available. AmpliTaq.TM. is a commercially available genetically engineered version of Taq DNA polymerase and is substantially equivalent to the native form. Perkin Elmer Cetus; Saiki and Gelfand, Amplifications (Perkin Elmer Cetus), 1, 4 (1989). Also commercially available is a truncated gene product, the Stoffel fragment, that expresses an enzyme lacking the 5'-3' exonuclease activity and having much lower unit activity, probably due to decreased processivity and increased mutagenesis. Barnes, supra. Gelfand and Abramson (PCT International Publication No. WO 92/06200) disclosed a modified Taq polymerase having the same length as the native enzyme, but with highly attenuated 5'-3' exonuclease activity. The exonuclease activity is defeated by mutation in nucleotide 137 of the Taq polymerase gene, wherein the mutation is G to A, resulting in a change in amino acid 46 of the enzyme from Gly to Asp. This enzyme is reported as having the same polymerase activity, processivity and extension rate as the native enzyme.